The objective of this project is to develop approaches that can be used for gene therapy f sickle cell disease and beta thalassemia. This will be attempted with development of vectors which will transfer the gamma globin genes into the patient' stem cells or vectors that will transfer transcriptional factors which can activate the normal gamma globin genes of the patients. We propose to 1) investigate elements that will pro protect the transferred genes from negative effects of the position of integration into the stem cell genome. Specifically, test whether the border elements of the chicken beta locus domain function as insulators in mammalian cells; search for insulator elements of the human beta globin locus; investigate putative insulators from other loci; test these elements in vivo following transplantations of transduced murine hemopoietic stem cells. 2) Develop optimal globin gene expression cassettes for viral vectors. Specifically, improve the expression cassettes we have already developed, investigate the structure-function relationships of the beta locus control region (LCR) and use this knowledge to produce optimal LCR cassettes; produce viral vectors carrying the optimal globin gene expression cassettes and test their function following stem cell transduction in normal mice and in thalassemic or sickle cell mice. 3) Test the feasibility of transactivation gene therapy of the beta chain hemoglobinopathies using the two new transcription activators of gamma and epsilon gene expression we have cloned; test whether their over- expression affects the normal murine hematopoiesis; investigate whether they can transactivate the human globin genes in transgenic mice carrying the human beta locus YACs or muLCR/Agamma gene recombinants; and test whether stem cell transfer of viral vectors carrying the genes codes for these transcriptional factors cure the cellular defects of the beta thalassemic or sickle cell mice.